Hapten-carrier conjugates for treating and preventing nicotine addiction

ABSTRACT

Novel hapten-carrier conjugates are capable of inducing the production of antibodies, in vivo, that specifically bind to nicotine. These conjugates comprise a nicotine hapten conjugated to an immunogenic carrier protein. The novel conjugates preserve the chirality of nicotine in its native (S)-(−) state, and have good stability properties. The conjugates are useful in formulating vaccines for active immunization, that are used to prevent and treat nicotine addiction. The antibodies raised in response to the nicotine hapten-carrier conjugate are used for passive immunization. These antibodies are administered for prevention and treatment of nicotine addiction.

FIELD OF THE INVENTION

The present invention relates to treatment and prevention of nicotineaddiction. In particular, the invention relates to novel hapten-carrierconjugates which are capable of inducing the production of antibodies.Such antibodies are capable of specifically binding to nicotine.Furthermore, the present invention envisages preventing or treatingnicotine addiction by administering a nicotine-carrier conjugate in apharmaceutically-acceptable formulation. The present invention alsocontemplates using the antibodies raised in response to thehapten-carrier conjugate for the prevention and treatment of nicotineaddiction.

BACKGROUND OF THE INVENTION

Smoking of cigarettes, cigars, and pipes is a prevalent problem in theUnited States and worldwide. Smoking tobacco and smokeless tobacco arerich in nicotine, which is a known addictive substance. Nicotine is analkaloid derived from the tobacco plant that is responsible forsmoking's psychoactive and addictive effects. Nicotine is formed of tworings linked together by a single bond: an aromatic six-membered ring(pyridine) and an aliphatic five-membered ring (pyrrolidine). Thepyrrolidine is N-methylated and linked through its carbon-2 to thecarbon-3 of pyridine. Thus, the carbon-2 is chiral, and there isvirtually free rotation around the single bond linking the two rings. Ithas been established that the absolute configuration of carbon-2 is S.Thus, the natural configuration of nicotine is (S)-(−)-nicotine.

Nicotine use is widespread due to the easy availability of cigarettes,cigars, pipes and smokeless tobacco. According to the U.S. Department ofHealth and Human Services, cigarette smoking is the single leading causeof preventable death in the United States. See also McGinnis et al., J.Am. Med. Assoc., 270, 2207–2211 (1993). Exposure to second hand smokealso has been reported to have serious detrimental health effects,including exacerbation of asthma.

Even though the addictive nature of nicotine is well known, cigarettesmoking is prevalent. Peak levels of nicotine in the blood, about 25 to50 nanograms/ml, are achieved within 10–15 minutes of smoking acigarette. In humans, smoking a cigarette results in arterial nicotineconcentrations being 10-fold higher than venous nicotine concentrationsbecause nicotine is rapidly delivered from the lungs to the heart (seeHenningfield (1993) Drug Alcohol Depend. 33:23–29). This results in arapid delivery of high arterial concentrations of nicotine to the brain.Once nicotine crosses the blood-brain barrier, evidence suggests that itbinds to cholinergic receptors, which are normally activated by theneurotransmitter acetylcholine, which is involved in respiration,maintenance of heart rate, memory, alertness and muscle movement. Whennicotine binds to these receptors, it can affect normal brain function,by triggering the release of other neurotransmitters, such as dopamine.Dopamine is found in the brain in regions involved in emotion,motivation, and feelings of pleasure. It is the release ofneurotransmitters, especially dopamine, that is responsible for thetobacco user's addiction to nicotine or other intake of nicotine.

Due to the significant adverse effects of smoking on health, smokersoften try to quit. However, the addictive nature of nicotine and theavailability of cigarettes add to the continued dependence on nicotineand high failure rate of those who try to quit. Withdrawal symptoms areunpleasant, and are relieved by smoking.

Many therapies for nicotine addiction have been developed, but arelargely ineffective. The two most popular therapies remain the nicotinetransdermal patch and nicotine incorporated into chewing gum. Thesetherapies, termed “nicotine replacement therapies” (NRT), replace theamount of nicotine which the user previously received from smoking andact to wean the user off nicotine. However, certain drawbacks are seenwith this type of therapy. Particularly, there is low penetration ofnicotine into the bloodstream and therefore an increased desire tosmoke. Problems such as mouth irritation, jaw soreness, nausea, havebeen associated with use of nicotine chewing gum. Problems such as skinirritations, sleep disturbance, and nervousness have been associatedwith use of nicotine transdermal patches.

Therefore, an alternative methodology for treating nicotine addiction isneeded. The literature recognizes this need and there have been severalattempts to provide a methodology for treating nicotine addiction. Oneof the methods involves the administration of antibodies which have beenraised in response to nicotine. However, low molecular weightsubstances, called haptens, are known to be unable to trigger an immuneresponse in host animals. Nicotine is no exception, and as a smallmolecule it is not immunogenic. To elicit an antibody response to ahapten, it typically is covalently bound to a carrier protein, and thecomplex will elicit the production of antibodies that recognize thehapten.

For example, cotinine 4′-carboxylic acid, when bound covalently tokeyhole limpet hemocyanin (KLH) was used to generate antibodies to thenicotine metabolite cotinine. Those antibodies were used to determinethe presence of cotinine in physiological fluids. See Bjerke et al. J.Immunol. Methods, 96, 239–246 (1987).

Other nicotine antibodies were prepared by Castro et al., (Eur. J.Biochem., 104, 331–340 (1980)). Castro et al. prepared nicotine haptens,conjugated to bovine serum albumin (BSA), with the carrier proteinconjugated via a linker at the 6-position of nicotine. Castro et al.prepared additional nicotine conjugates of BSA which were injected intomammals to raise antibodies. In another publication, Castro et al. inBiochem. Biophys. Res. Commun. 67, 583–589 (1975) disclose two nicotinealbumin conjugates: N-succinyl-6-amino-(±)-nicotine-BSA and6-(σ-aminocapramido)-(±)-nicotine-BSA. In this 1975 publication, Castroet al. also used antibodies to nicotine carrier conjugate,6-(σ-aminocapramido)-(±)-nicotine-BSA, to determine the levels ofnicotine in blood and urine, see Res. Commun Chem. Path. Pharm. 51,393–404 (1986).

Swain et al. (WO 98/14216) disclose nicotine carrier conjugates whereinthe hapten is conjugated at the 1, 2, 4, 5, 6, or 1′ position of thenicotine. Hieda et al. have shown that animals immunized with6-(carboxymethylureido)-(±)-nicotine, which was linked to keyhole limpethemocyanin, produced antibodies specific to nicotine. J. Pharm. andExper. Thera. 283, 1076–1081 (1997). Langone et al. prepared the haptenderivative, O-succinyl-3′-hydroxymethyl-nicotine, see Biochemistry, 12,5025–5030, and used the antibodies to this hapten carrier conjugate inradioimmunoassays. See Methods in Enzymology, 84, 628–635 (1982). Theconjugate produced by Langone is susceptible to hydrolysis.Additionally, Abad et al. in Anal. Chem., 65, 3227–3231 (1993) describeconjugating 3′-(hydroxymethyl) nicotine hemisuccinate to bovine serumalbumin to produce antibodies to nicotine in order to be able to measurenicotine content in smoke condensate of cigarettes in an ELISA assay.

Therefore, the prior art does not teach a stable nicotine-carrierconjugate that preserves the chiral nature of the nicotine hapten, andthat links the hapten to the carrier in a way that conserves the natureof the nicotine epitope(s). Moreover, the art does not teach or suggestmethods of preventing and treating nicotine addiction by using suchconjugates. Seeman in Heterocycles, 22, 165–193, (1984) disclosesresults of a study of the conformational analysis and chemicalreactivity of nicotine.

SUMMARY OF THE INVENTION

In response to the demand for a more effective methodology for treatingnicotine addiction, it is one object of the,present invention to providenovel nicotine-carrier conjugates that are stable, comprise nicotine inits natural (S)-(−) formation, and employ a nicotine-carrier linkagethat preserves the nature of the nicotine epitope(s), and the relativeorientation of the two rings of the nicotine molecule. Both rings ofnicotine, and their relative orientation, are believed to be essentialfor the recognition by antibody of nicotine in solution. Such conjugatesare capable of stimulating the production of antibodies that are capableof specifically binding to nicotine. Using the inventive conjugates, theinventors have raised serum nicotine levels, and decreased brainnicotine levels, in mammals. Additionally, using the conjugates of theinvention, the inventors also have prevented nicotine-induced changes inblood pressure, and locomotor effects.

In another object of the present invention is provided a method oftreating nicotine addiction by administering a conjugate of theinvention to a patient addicted to nicotine thereby generateanti-nicotine antibodies in that patient. Thus, when the patient smokes(or uses chewing tobacco), the nicotine from these products will bebound by the anti-nicotine antibodies in the blood, preventing thenicotine from crossing the blood-brain barrier, hence eliminating thenicotine-induced alterations in brain chemistry, which is the source ofnicotine-addiction. In this regard, it is important that thenicotine-carrier conjugate elicit the production of antibodies that willrecognize the native nicotine molecule. As described above, the novelnicotine-carrier conjugates of the invention preserve the chirality andthe epitope(s) of naturally-occurring nicotine.

The inventors do not intend to be bound by any particular theory as tohow the nicotine conjugates, and the antibodies produced in response tosuch conjugates, inhibit the effects of nicotine ingested by mammals. Inaddition to preventing nicotine from crossing the blood brain barrier,the antibodies also may prevent nicotine from binding to other receptorsin the peripheral nervous system by simple steric blockage.

These objects can be achieved by providing a hapten-carrier conjugate offormula (I):

wherein m is 1 to 2500, n is 0 to 12, y is 1 to 12, X is selected fromthe group consisting of NH—CO, CO—NH, CO—NH—NH, NH—NH—CO, NH—CO—NH,CO—NH—NH—CO, and S—S; Y is selected from the group consisting of NH—CO,CO—NH, CO—NH—NH, NH—NH—CO, NH—CO—NH, CO—NH—NH—CO, and S—S, and the—(CH₂)_(n)—X—(CH₂)_(y)—Y— moiety is bonded to the 3′, 4′ or 5′ position.In a preferred embodiment of the hapten carrier conjugate, m is 11 to17, n is 1, y is 2, X is NH—CO, Y is CO—NH, the carrier protein isexoprotein A and the —(CH₂)_(n)—X—(CH₂)_(y)—Y— moiety is bonded to the3′ position. In another preferred embodiment of the hapten-carrierconjugate, m is 11 to 17, n is 1, y is 2, X is NH—CO, Y is CO—NH, thecarrier protein is exoprotein A and the —(CH₂)_(n)—X—(CH₂)_(y)—Y— moietyis bonded to the 4′ position. In a further preferred embodiment of thehapten-carrier conjugate, m is 11 to 17, n is 1, y is 2, X is NH—CO, Yis CO—NH, the carrier protein is exoprotein A and the—(CH₂)_(n)—X—(CH₂)_(y)—Y— moiety is bonded to the 5′ position. In anadditionally preferred embodiment, m is selected from the groupconsisting of 1 to 20 and 1 to 200.

The above objects also be achieved by providing a hapten-carrierconjugate of formula (III):

wherein n is 0 to 12, j is 1 to 1000, k is 1 to 20, and E is an aminoacid-containing matrix. In a preferred embodiment, the matrix ispoly-L-glutamic acid.

The objects can also be achieved by providing an antibody which isproduced in response to the hapten-carrier conjugate of Formula (I). Inan additional embodiment, the antibody is a functional fragment. In apreferred embodiment, the antibody is a monoclonal antibody. In anadditional embodiment of the invention, the antibody is polyclonal.

The objects can also be achieved by providing an antibody which isproduced in response to the hapten-carrier conjugate of Formula (III).In an additional embodiment, the antibody is a functional fragment. In apreferred embodiment, the antibody is a monoclonal antibody. In anadditional embodiment of the invention, the antibody is polyclonal.

The objects can be achieved by providing a method of treating orpreventing nicotine addiction in a patient in need of such treatmentcomprising administering a therapeutically effective amount of thehapten-carrier conjugate of Formula (I) or (III). Alternatively, theobjects can be achieved by providing a method treating or preventingnicotine addiction in a patient in need of such treatment comprisingadministering a therapeutically effective amount of antibody raised inresponse to the hapten-carrier conjugates of Formula (I) or (III).

Additionally, the objects can be achieved by providing a vaccinecomposition which comprises the hapten carrier conjugate of Formula (I)or Formula (III). In addition the vaccine can further comprise anadditional therapeutic compound for treating nicotine addiction.

The objects also can be achieved by providing a process for producing anantibody, comprising immunizing a host mammal with a hapten-carrierconjugate of Formula (I) or (III). In a preferred embodiment, theantibody produced is a monoclonal antibody. In an additional embodimentthe antibody is polyclonal.

Additional objects can be achieved by providing a kit for determiningthe presence of nicotine in a sample, comprising an antibody of raisedin response to the hapten-carrier conjugate of Formula (I) or Formula(III).

These objects and others apparent to those skilled in the art have beenachieved by the invention described below in the detailed descriptionand appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart that shows the effect of active immunization, with a3′AMNic-Suc-rEPA conjugate vaccine, on nicotine blood serum levels inrats, following a singe injection of nicotine. Nicotine serum levels, 3and 10 minutes after nicotine injection, are shown.

FIG. 2 shows the effect of passive immunization, with antibodies against3′-AMNic-Suc-rEPA, on nicotine levels in blood and brain of rats. Ratswere treated with 12.5, 25 and 50 mg of antibody.

FIG. 3 shows the effects of passive immunization, with antibodiesagainst 3′-AMNic-Suc-rEPA, on nicotine levels in blood serum and brain,in rats. Nicotine levels were measured 30 minutes and 1 day afterantibody administration and 3 minutes for nicotine injection.

FIG. 4 shows the effect of passive immunization, with antibodies against3′-AMNic-Suc-rEPA, on nicotine blood serum levels, in rats receivingmultiple doses of nicotine.

FIG. 5 shows the effects of passive immunization, with antibodiesagainst 3′-AMNic-Suc-rEPA, on nicotine levels in rat brain, in ratsreceiving multiple doses of nicotine.

FIG. 6 shows the effects of passive immunization, with antibodiesagainst 3′-AMNic-Suc-rEPA, on nicotine-induced locomoter effects, inrats.

FIG. 7 shows the effects of passive immunization, with antibodiesagainst 3′-AMNic-Suc-rEPA, on nicotine-induced increase in systolicblood pressure. The Figure shows that the increasing amounts of antibodyincreases the effectiveness of the antibodies in decreasing thenicotine-increase in blood pressure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a nicotine hapten carrier conjugate fortreating addiction to nicotine. The nicotine hapten-carrier conjugate isof formula (I):

wherein m is 1 to 2,500; n is 0 to 12; y is 1 to 12; X is selected fromthe group consisting of NH—CO, CO—NH, CO—NH—NH, NH—NH—CO, NH—CO—NH,CO—NH—NH—CO and S—S; Y is selected from the group consisting of NH—CO,CO—NH, CO—NH—NH, NH—NH—CO, NH—CO—NH, CO—NH—NH—CO and S—S; the carrierprotein is any suitable immunogenic protein or polypeptide. Preferablythe carrier protein may comprise a T-cell epitope, and the—(CH₂)_(n)—X—(CH₂)_(y)—Y— moiety is bonded to the 3′, 4′ or 5′ positionof the nicotine molecule.

In formula (I), m is preferably 1 to 200. In another preferredembodiment, m is 1 to 20. In a particularly preferred embodiment, m is11 to 17. In another preferred embodiment, X is selected from the groupconsisting of NH—CO, CO—NH, CO—NH—NH, NH—NH—CO, NH—CO—NH, andCO—NH—NH—CO.

If m is more than one, the moiety in brackets is attached m times todifferent points of attachment in the carrier protein. For example, ifm=2, then formula (I) would be:

Because antibodies cannot be raised in response to nicotine itself, thepresent inventors have developed a nicotine hapten which is derivatizedat the 3′, 4′, or 5′ position of nicotine. This moiety is bound to acarrier protein to yield a hapten carrier conjugate, which will raiseantibodies against the nicotine moiety, when it is injected into asuitable host mammal. In this regard, in order for a pharmaceuticalcomposition comprising the hapten carrier conjugate to induce theproduction of antibodies when administered to a mammal, the carrierprotein must be immunogenic. Preferably, it will comprise a Tcell-epitope. Thus, when the carrier protein is conjugated to thenicotine hapten, and subsequently is administered to a mammal, themammal produces, or “raises” antibodies in response to the nicotinehapten.

Haptens and Derivatization

The term “hapten” as used in the present invention refers to alow-molecular weight organic compound that is not capable of elicitingan immune response by itself but will elicit an immune response onceattached to a carrier molecule. In a preferred embodiment, the hapten isattached to the carrier via a linker. A hapten of the present inventionis a nicotine derivative. This nicotine hapten contains a reactivefunctional group, to which the carrier can be attached directly, or viaa linker, or via a matrix, or via a linker and a matrix. Preferably, thenicotine hapten is attached to the carrier protein via an amide ordisulfide bond. Amide and disulfide bonds have the desirable property ofstability. Because the hapten-carrier conjugates of the invention willbe used as vaccines, it is important that the conjugates are stable, toprolong the shelf life of the vaccine.

In a preferred embodiment of the present invention, the nicotine haptenis represented by formula (II):

wherein n is 0 to 12 and Z is NH₂, COOH, CHO or SH and —(CH₂)_(n)—Z canbe bonded to the 3′, 4′ or 5′ position. The Z moiety is capable ofbinding to a carrier, directly or via a linker. The carrier-haptenconjugate will induce the production of antibodies upon its introductioninto the body of a patient or an animal.

In a particularly preferred embodiment, the nicotine hapten is of thefollowing formula (3′-aminomethyl nicotine):

1. Direct Conjugates

To make a “direct conjugate,” a single nicotine hapten is directlyattached to a carrier, with or without a linker. For example, a singlenicotine hapten can be attached to each available amine group on thecarrier. General methods for directly conjugating haptens to carrierproteins, using a homobifunctional or a heterobifunctional cross-linkerare described, for example, by G. T. Hermanson in BioconjugateTechniques, Academic Press (1996) and Dick and Beurret in ConjugateVaccines. Contribu. Microbiol. Immunol. Karger, Basal (1989) vol. 10,48–114. With direct conjugation using bifunctional crosslinkers, themolar ratio of hapten to protein is limited by the number of functionalgroups available on the protein for the specific conjugation chemistry.For example, with a carrier protein possessing n number of lysinemoieties, there will be, theoretically, n+1 primary amines (includingthe terminal amino) available for reaction with the linker's carboxylicgroup. Thus, using this direct conjugation procedure the product will belimited to having n+1 amido bonds formed, i.e., a maximum of n+1 haptensattached.

The skilled artisan will recognize that depending on the concentrationof the reactants used to conjugate the nicotine hapten to the carrierprotein, and the nature of the carrier protein, the ratio of hapten tocarrier will vary. Also, within a given preparation of nicotine-carrierconjugate, there will be variation in the hapten/carrier ratio of eachindividual conjugate. For example, exoprotein A has, in theory, 15amines available for conjugation with hapten. However, the inventorsdetermined that when 3′aminomethyl-succinyl-nicotine was conjugated tothis protein, a range of 11–17 nicotine haptens were attached to eachexoprotein A carrier, in a single preparation of conjugate. This rangewas experimentally determined using gas filtration chromatography andmeasuring the increase in UV absorbance at 260 nm. 17 nicotines wereattached to some carriers because the nicotine hapten can attach tonon-amine moieties on the carrier. Examples of non-amine moieties towhich the hapten can attach include, but are not limited to, —SH and —OHmoieties. However, the incidence of these side reactions is low.

2. Matrix Conjugates

To circumvent the limitations on the number of haptens that can beattached to carrier using direct conjugation, an amino acid “matrix” canbe used. The term “matrix” denotes an amino acid, a peptide, dipeptide,or a polypeptide, including oligomeric and polymeric polypeptides. Amatrix also may be a linear or branched polypeptide. Examples of aminoacids that may be used to form a matrix include, but are not limited to,aspartic acid, lysine, cysteine, and L-glutamic acid. Such matrixmaterials may be formulated into polymers, such as poly-L-glutamic acid.When an amino acid such as cysteine is used, the thiol group isprotected, thereby permitting the hapten to be linked to the carboxylicgroup of the amino acid. One skilled in the art would be well familiarwith types of protecting groups and means of attaching protecting groupsto amino acid functionalities. For a discussion, see Green, PROTECTIVEGROUPS IN ORGANIC CHEMISTRY, John Wiley & Sons, New York, 1991.

A suitable matrix possesses an appropriate functional group and isloaded with two or more haptens. Thus, in another preferred embodimentof the invention, the nicotine-substituted matrix is conjugated to thecarrier protein to increase the hapten to carrier molar ratio in thehapten-carrier conjugate. The matrix plays a double role, first, as asupport for a large number of haptens and, second, as a cross linker.The nicotine substituted matrix conjugated to a carrier protein isrepresented by formula (II):

wherein n is 0 to 12, j is 1 to 1000, k is 1 to 20 E is an aminoacid-containing matrix to which a hapten can be bonded, and the carrierprotein is any suitable protein or polypeptide comprising a T-cellepitope. The amino acid-containing matrix E may be an amino acid, apeptide, dipeptide, or a polypeptide, including oligomeric as well aspolymeric polypeptides. The matrix comprises one or more amino acidsthat include, but are not limited to, aspartic acid, lysine, cysteine,and poly-L-glutamic acid. In a preferred embodiment, j is 1 to 200, andin another preferred embodiment, j is 1 to 4.

Matrix-carrier conjugates are capable of forming multimeric “lattices.”Such a lattice is represented in the figure below. The term “lattice” isused to denote a covalently-linked complex, comprising multiplematrices, haptens, linkers and carrier proteins, all of which arecovalently linked together. Because the nicotine-substituted matrixcomprises multiple nicotine moieties available for conjugation withcarrier, a lattice comprising multiple carriers, and multiplenicotine-substituted matrices, can be formed. A simplifiedrepresentation of a portion of such a lattice is represented as follows:

The skilled artisan will recognize that a lattice according to theinvention comprises a hapten carrier conjugate of Formula (III).

This conjugation method employing a matrix offers flexibility andcontrol over hapten to protein molar ratios regardless of the number offunctional groups available for conjugation on the protein. This isparticularly useful when a specific carrier protein has been used andwhen an optimal ratio needs to be obtained in order to achieve higherimmunogenicity of the conjugate. While it is not necessary to use anwhen using a matrix, such a linker can be used. To use a linker withthis embodiment, the nicotine substituted matrix is reacted with anactive linker compound. For example, ADH, adipic acid dihydrazide, canbe used as a linker with the matrix conjugates.

Carrier Proteins

Once the nicotine hapten has been prepared, it is then conjugated to acarrier protein which will be used to raise antibodies to the nicotinecarrier conjugate. The carrier protein used in the present inventivenicotine carrier conjugate is represented by

in formulae (I) and (III) and encompasses any suitable immunogenicprotein or polypeptide. An “immunogenic” molecule is one that is capableof eliciting an immune response. Preferably, the carrier protein willcomprise a T-cell epitope. Also encompassed by the representation of a“carrier protein” are MAPs or multi-antigenic peptides, which arebranched peptides. By using a MAP, hapten density and valency aremaximized because of multiple branched amino acid residues. Examples ofamino acids that can be used to form a MAP include, but are not limitedto, lysine.

A carrier protein of the instant invention comprises a moleculecontaining at least one T cell epitope which is capable of stimulatingthe T cells of the subject, which subsequently induces B cells toproduce antibodies against the entire hapten-carrier conjugate molecule.The term “epitope” as used in describing this invention, includes anydeterminant on an antigen that is responsible for its specificinteraction with an antibody molecule. Epitopic determinants usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and have specific three dimensionalstructural characteristics as well as specific charge characteristics.It is believed that to have immunogenic properties, a protein orpolypeptide must be capable of stimulating T-cells. However, it ispossible that a carrier protein that lacks a T-cell epitope may also beimmunogenic.

By selecting a carrier protein which is known to elicit a strongimmunogenic response, a diverse population of patients can be treated bythe inventive hapten-carrier conjugates. The carrier protein must besufficiently foreign to elicit a strong immune response to the vaccine.Typically, the carrier protein used preferably would be a large moleculethat is capable of imparting immunogenicity to a covalently-linkedhapten. A particularly preferred carrier protein is one that isinherently highly immunogenic. Thus a carrier protein that has a highdegree of immunogenicity and is able to maximize antibody production tothe hapten is highly desirable.

Both bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) havecommonly been used as carriers in the development of conjugate vaccineswhen experimenting with animals. However, these proteins may not besuitable for human use. Proteins which have been used in the preparationof therapeutic conjugate vaccines include, but are not limited to, anumber of toxins of pathogenic bacteria and their toxoids. Examplesinclude diphtheria and tetanus toxins and their medically acceptablecorresponding toxoids. Other candidates are proteins antigenicallysimilar to bacterial toxins referred to as cross-reacting materials(CRMs).

In the preparation of nicotine conjugate pharmaceutical compositions,recombinant Pseudomonas aeruginosa exoprotein A (rEPA) may be used as acarrier protein because its structure and biological activities havebeen well characterized. Moreover, this recombinant protein has beensuccessfully and safely used in humans in the Staphylococcus aureuscapsular polysaccharide conjugate vaccines by the National Institutes ofHealth and by the present inventors. Fattom et al., Infect Immun. 611023–1032 (1993). This protein has been identified as a suitable proteincarrier because the intrinsic enzymatic activity of the native exotoxinhas been eliminated due to an amino acid deletion at position 553. As aresult, rEPA has the same immunological profile as the native exotoxin A(ETA), but does not possess the hepatotoxic properties of the nativeETA. As used in this application, “exoprotein A” refers to a modified,non-hepatotoxic, ETA. On example of such an exoprotein A has an aminoacid deletion at position 553.

Conjugation of Hapten to Carrier Protein

There are a large number of functional groups which can be used in orderto facilitate the linking or conjugation of a carrier to a smallmolecule, such as a hapten. These include functional moieties such ascarboxylic acids, anhydrides, mixed anhydrides, acyl halides, acylazides, alkyl halides, N-maleimides, imino esters, isocyanates, amines,thiols, and isothiocyanates and others known to the skilled artisan.These moieties are capable of forming a covalent bond with a reactivegroup of a protein molecule. Depending upon the functional moiety used,the reactive group may be the E amino group of a lysine residue or athiol group, on a carrier protein or a modified carrier protein moleculewhich, when reacted, results in amide, amine, thioether, amidine urea orthiourea bond formation. One skilled in the art would recognize thatother suitable activating groups and conjugation techniques can be used.See, for example, Wong, Chemistry of Protein Conjugation andCross-Linking, CRC Press, Inc. (1991). See also Hermanson, BIOCONJUGATETECHNIQUES, Academic Press: 1996 and Dick and Beurret in ConjugateVaccines. Contribu. Microbiol. Immunol., Karger, Basal (1989) vol. 10,48–114.

Linear linker moieties are preferred, over cyclic or branched linkers,for conjugation of haptens to carrier proteins. A preferred linker is asuccinyl moiety. However, a linker may be a cyclic structure as well asa linear moiety. Another example of a linker is ADH.

Thus, the nicotine hapten-carrier conjugates of the present inventionare prepared by reacting one or more haptens with a carrier protein toyield a hapten carrier conjugate which is capable of stimulating Tcells, leading to T cell proliferation and release of mediators whichactivate specific B cells to stimulate antibody production in responseto the immunogenic hapten carrier conjugate. Certain antibodies raisedin response to the hapten carrier conjugate will be specific to thehapten portion of the hapten-carrier conjugate. The present inventioncontemplates the use of various suitable combinations of haptens withcarrier proteins for use in the treatment of nicotine addiction.

Monoclonal and Polyclonal Antibodies

Techniques for making monoclonal antibodies are well-known in the art.Monoclonal antibodies can be obtained by injecting mice with acomposition comprising the nicotine hapten carrier conjugate,subsequently verifying the presence of antibody production by removing aserum sample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to thehapten-carrier conjugate, culturing the clones that produce antibodiesto the antigen, and isolating the antibodies from the hybridomacultures.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1–2.7.12 and pages 2.9.1–2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79–104 (The Humana Press, Inc. 1992).

Techniques for preparing polyclonal antibodies also are well-known inthe art. Polyclonal antibodies are prepared according to standardtechniques known in the art. To prepare a polyclonal antibody, an animalis injected with the immunogenic material and antibody rich serum iscollected which contains therein a mixture of antibodies that aredirected against numerous epitopes of the immunogen that was injected.Suitable host mammals for the production of antibodies include, but arenot limited to, humans, rats, mice, rabbits, and goats.

In accordance with the present invention, functional antibody fragmentsalso can be utilized. The fragments are produced by methods that includedigestion with enzymes such as pepsin or papain and/or cleavage ofdisulfide bonds by chemical reduction. Alternatively, antibody fragmentsencompassed by the present invention can be synthesized using anautomated peptide synthesizer such as those supplied commercially byApplied Biosystems, Multiple Peptide Systems and others, or they may beproduced manually, using techniques well known in the art. See Geysen etal., J. Immunol. Methods 102: 259 (1978). Direct determination of theamino acid sequences of the variable regions of the heavy and lightchains of the monoclonal antibodies according to the invention can becarried out using conventional techniques.

A fragment according to the present invention can be an Fv fragment. AnFv fragment of an antibody is made up of the variable region of theheavy chain (Vh) of an antibody and the variable region of the lightchain of an antibody (Vl). Proteolytic cleavage of an antibody canproduce double chain Fv fragments in which the Vh and Vl regions remainnon-covalently associated and retain antigen binding capacity. Fvfragments also include recombinant single chain antibody molecules inwhich the light and heavy chain variable regions are connected by apeptide linker. See Skerra, et al. Science, 240, 1038–41 (1988).Antibody fragments according to the invention also include Fab, Fab′,F(ab)₂, and F(ab′)₂, which lack the Fc fragment of an intact antibody.

Therapeutic Methods

Because nicotine exerts many of its significant effects after it crossesthe blood brain barrier, the present invention encompasses therapeuticmethods that prevent nicotine from crossing the blood brain barrier. Inparticular, administration of a nicotine hapten-carrier conjugate to apatient will generate antibodies against nicotine, in the bloodstream ofthe patient. Alternatively, anti-nicotine antibodies generated outsidethe body of the patient to be treated, in a suitable host mammal, can beadministered to a patient. If the patient smokes, the nicotine in hisblood will be bound by the circulating anti-nicotine antibodies,preventing the nicotine from reaching the brain. Therefore, theantibodies will prevent the physiological and psychological effects ofnicotine that originate in the brain. Because the smoker will experiencea lessening or cessation of these effects, he/she will lose the desireto smoke. The same therapeutic effects are expected if a patient usessmokeless tobacco, after being immunized with a nicotine hapten-carrierconjugate of the invention. Additionally, the conjugates and antibodiesof the invention may exert their effects by affecting the ability ofnicotine to stimulate the peripheral nervous system.

As discussed above, the novel nicotine-carrier conjugates of theinvention preserve the native chirality and structure of the nicotinemolecule. In particular, the nicotine moiety of these conjugates has the(S)-(−) configuration. Therefore, the antibodies produced in response tosuch a conjugate will be specific to the native form of nicotine, andwill be the most effective in specifically binding to nicotine that isinhaled from smoking or absorbed from smokeless tobacco, and ininhibiting the effects of this ingested nicotine. Additionally, theinventive conjugates are chemically stable, and stability is critical toproducing a vaccine having a long shelf life.

The present vaccine composition can be used in combination withcompounds or other therapies that are useful in the treatment ofaddiction. This includes administration of compounds which include, butare not limited to, anti-depressant drugs such as Zyban and Prozac.

1. Administration of a Nicotine Hapten-Carrier Conjugate

The conjugates of the invention are suitable for treating and preventingnicotine addiction. For treating nicotine addiction, a nicotine-carrierconjugate of the invention is administered to a patient suffering fromnicotine addiction. For preventing nicotine addiction, patients at riskfor developing nicotine addiction, such as teenagers, are treated with aconjugate according to the invention. Direct administration of theconjugate to a patient is called “active immunization.”

A vaccine composition of the present invention comprises at least onenicotine hapten-carrier conjugate in an amount sufficient to elicit animmune response thereto. The nicotine hapten carrier conjugate iscapable of remaining in vivo at a concentration sufficient to be activeagainst subsequent intake of nicotine.

Initial vaccination with the nicotine hapten carrier conjugate of thepresent invention creates high titers of antibodies that are specific tonicotine. The therapeutically effective amount of a conjugate which isadministered to a patient in need of treatment for nicotine addiction isreadily determined by the skilled artisan. Suitable dosage ranges are1–1000 μg/dose. It generally takes a patient one to several weeks togenerate antibodies against a foreign antigen. The production ofantibodies in a patient's blood can be monitored by using techniquesthat are well-known to the skilled artisan, such as ELISA,radioimmunoassay, and Western blotting methods. Therapeuticeffectiveness also can be monitored by assessing various physicaleffects of nicotine, such as blood pressure.

As described in detail below, the inventive nicotine hapten-carrierconjugates can be processed to afford a composition which can be readilyadministered to a patient. The preferred modes of administration includebut are not limited to intranasal, intratracheal, oral, dermal,transmucosal subcutaneous injection and intravenous injection. Theskilled artisan will recognize that the initial injection may befollowed by subsequent administration of one or more “boosters” ofconjugate. Such a booster will increase the production of antibodiesagainst the nicotine hapten carrier conjugate of the invention.

The vaccine compositions of the present invention may contain at leastone adjuvant. The adjuvant used in the present invention will beselected so that the effect of the carrier protein is not inhibited.Adjuvants used in the present invention are those which arephysiologically acceptable to humans, these include, but are not limitedto, alum, QS-21, saponin and MPLA (monophosphoryl lipid A).

The vaccine compositions of the present invention may optionally containone or more pharmaceutically acceptable excipients. The excipientsuseful in the present include sterile water, salt solutions such assaline, sodium phosphate, sodium chloride, alcohol, gum arabic,vegetable oils, benzyl alcohols, polyethylene glycol, gelatin, mannitol,carbohydrates, magnesium stearate, viscous paraffin, fatty acid esters,hydroxy methyl cellulose and buffers. Of course, any additionalexcipients known to the skilled artisan are useful in the presentinvention.

The hapten-carrier conjugates of the present invention, in order to beadministered to a patient in need of treatment or prevention of nicotineaddiction, are incorporated into a pharmaceutical composition. When thecomposition containing the hapten-carrier conjugate is to be used forinjection, it is preferable to solubilize the hapten-carrier conjugatein an aqueous, saline solution at a pharmaceutically acceptable pH.However, it is possible to use an injectable suspension of thehapten-carrier conjugate. In addition to the usual pharmaceuticallyacceptable excipients, the composition may contain optional componentsto ensure purity, enhance bioavailability and/or increase penetration.

Additionally, the vaccine composition may optionally contain at leastone auxiliary agent, such as dispersion media, coatings, microspheres,liposomes, microcapsules, lipids, surfactants, lubricants, preservativesand stabilizers. Of course, the any additional auxiliary agents known tothe skilled artisan are useful in the present invention. Also usefulherein are any agents which act to synergize the effect of the presentvaccine composition.

The pharmaceutical composition of the present invention is sterile andis sufficiently stable to withstand storage, distribution, and use.Additionally, the composition may contain additional components in orderto protect the composition from infestation with, and growth of,microorganisms. It is preferred that the composition is manufactured inthe form of a lyophilized powder which is to be reconstituted by apharmaceutically acceptable diluent just prior to administration.Methods of preparing sterile injectable solutions are well known to theskilled artisan and include, but are not limited to, vacuum drying,freeze-drying, and spin drying. These techniques yield a powder of theactive ingredient along with any additional excipient incorporated intothe pre-mix.

2. Administration of Antibodies Produced in Response to aNicotine-Carrier Conjugate

Passive immunization comprises administration of or exposure to apolyclonal antibody or monoclonal antibody which has been raised inresponse to a nicotine hapten carrier conjugate of the invention. Suchantibodies can be generated in animals or humans. Antibodies raised inresponse to a nicotine conjugate of the invention can be administered toprevent addiction to nicotine. For example, such antibodies can beadministered to people considered to be at risk for developing addictionto nicotine, such as teenagers. Antibodies also are suitable fortreating a patient addicted to nicotine. As discussed above, theantibodies will bind nicotine in the blood, and prevent nicotine fromcrossing the blood brain barrier. Antibodies raised by administration ofthe inventive hapten-carrier conjugate have a molecular weight range offrom about 150 kDa to about 1,000 kDa.

The therapeutically effective amount of a therapeutic antibody of theinvention which is administered to a patient in need of treatment fornicotine addiction is readily determined by the skilled artisan.Suitable dosage ranges are 1–1000 μg/dose.

A therapeutic composition of the present invention comprises at leastantibody produced in response to a nicotine-carrier conjugate of theinvention. These compositions of the present invention may optionallycontain one or more pharmaceutically acceptable excipients. Theexcipients useful in the present include sterile water, salt solutionssuch as saline, sodium phosphate, sodium chloride, alcohol, gum arabic,vegetable oils, benzyl alcohols, polyethylene glycol, gelatin, mannitol,carbohydrates, magnesium stearate, viscous paraffin, fatty acid esters,hydroxy methyl cellulose and buffers. Of course, any additionalexcipients known to the skilled artisan are useful in the presentinvention.

The antibodies of the present invention, in order to be administered toa patient in need of treatment or prevention of nicotine addiction, areincorporated into a pharmaceutical composition. When the compositioncontaining an antibody is to be used for injection, it is preferable tohave the antibody in an aqueous, saline solution at a pharmaceuticallyacceptable pH. However, it is possible to use an injectable suspensionof the antibody. In addition to the usual pharmaceutically acceptableexcipients, the composition may contain optional components to ensurepurity, enhance bioavailability and/or increase penetration.

A pharmaceutical composition comprising an antibody of the presentinvention is sterile and is sufficiently stable to withstand storage,distribution, and use. Additionally, the composition may containadditional components in order to protect the composition frominfestation with, and growth of, microorganisms. Methods of preparingsterile injectable solutions are well known to the skilled artisan andinclude, but are not limited to, vacuum drying, freeze-drying, and spindrying. These techniques yield a powder of the active ingredient alongwith any additional excipient incorporated into the pre-mix.

Kits Comprising Antibodies of the Invention

The antibodies of the present invention also are useful in preparing akit that can be used to detect and quantify nicotine levels in a sample.A kit according to the invention comprises a nicotine-specific antibodyaccording to the invention, in a suitable container. For aradioimmunoassay, the kit may also comprise labeled nicotine. Nicotinein a sample is detected by binding labeled nicotine to the antibody, andthen competing the labeled nicotine from the antibody with the sample tobe tested. An ELISA kit also would comprise an antibody according to theinvention. The ELISA may involve inhibition of antibody binding withknown amounts of nicotine compared to inhibition with a sample suspectedof containing nicotine. This would allow determination of unknownnicotine in a sample, by comparison of sample with the standardinhibition curve of known nicotine concentration. In another type ofELISA, a sample suspected of containing nicotine would be incubated witha microtiter plate that has been coated with a substance that will bindnicotine. The antibodies of the invention would be added, andenzyme-linked anti-antibody antibodies would be added to the plates.Addition of substrate would quantify the amount of nicotine bound to theplate.

The following examples are provided merely to further illustrate thepreparation and use of the present invention. The scope of the inventionis not limited to the following examples.

EXAMPLE 1 Synthesis of a Derivitized Nicotine Hapten (Substituted at the3′ Position)

The starting material for the synthesis of the hapten istrans-4′-carboxy-(−)-cotinine, available from commercial sources. Amodification of the procedure described by Cushman and Castagnoli, Jr.(1972) J. Org. Chem. 37(8):1268–1271 provides the alcohol,trans-3′-hydroxymethyl-(−)-nicotine, after methyl esterification of theacid followed by reduction of the ester. The alcohol is then sulfonatedand the sulfonate is displaced with an azido group, which is finallyreduced to an amine.

4 g of trans-4′-carboxy-(−)-cotinine are dissolved in 50 mL of asolution of 2 N sulfuric acid in dry methanol and stirred overnight atroom temperature. The resulting suspension is filtered through a WhatmanNo. 1 filter paper and added slowly to 100 mL of a saturated solution ofsodium bicarbonate. The ester is extracted with dichloromethane toafford 4.2 g of a pink oil after solvent evaporation.

A solution of 3.9 g of the ester in dry tetrahydrofuran (100 mL) isadded dropwise to a suspension of 4 equivalents of lithium aluminumhydride in dry tetrahydrofuran (70 mL) under dry argon. The suspensionis stirred for two hours at room temperature. Excess hydride isdestroyed by careful and controlled addition of water while cooling inan ice bath. The resulting white precipitate is filtered off and thefiltrate dried over sodium sulfate and concentrated under reducedpressure to afford 2.7 g of the alcohol as a yellow oil.

The alcohol (1.9 g) is dissolved in 20 mL of dichloromethane.Triethylamine (0.75 mL) and p-toluenesulfonyl chloride(1 g) are thensuccessively added to the solution. The orange solution is stirred for24 hours at room temperature. Precipitated triethylamine hydrochlorideis filtered off on a Celite bed and the filtrate is concentrated underreduced pressure to give a brown oil. The sulfonate is purified on asilica flash chromatography column eluted with 5% methanol indichloromethane to give 2.1 g of a yellow oil.

The sulfonate (1.8 g) is displaced using sodium azide (0.8 g) in 50 mLdimethylformamide for one hour at 80° C. After evaporation ofdimethylformamide under high vacuum, the residue is dissolved indichloromethane, washed with water and brine and dried over sodiumsulfate. After solvent evaporation, the azide (1.1 g) is obtained as abrownish oil.

The addition of the azide in dry tetrahydrofuran (20 mL) to a suspensionof lithium aluminum hydride in dry tetrahydrofuran (50 mL) readilyproduced the desired amine as monitored by thin layer chromatography.Proton and carbon nuclear magnetic resonance spectra of the purifiedamine corresponded to the expected structure.

EXAMPLE 2 Synthesis of a Derivitized Nicotine Hapten (Substituted at the4′ Position)

Introduction of a functionalized arm on position 4′ of nicotine can beachieved by enolate alkylation of cotinine followed by reduction of thealkylated product. Various alkylating agents can be used like anappropriately protected 3-bromo-propylamine. As examples,3-bromo-N-carbobenzyloxy-propylamine or N-(3-bromopropyl)-phtalamide canbe used. The amine protecting group will have to be removed afteralkylation and reduction and prior to conjugation to a carrier protein.Enolate alkylation of cyclic lactams (containing the pyrrolidinone ring)is well documented in the literature (see G. Helmchen et al. (1995)Steroselective Synthesis in Houben-Weyl-Methods of Organic Chemistry,Vol. E21a, 762–881, Thieme, Stuttgart, Germany, for a general review,and A. J. Meyers et al. (1997) J. Am. Chem. Soc., 119, 4564–4566, forsteric considerations of the reaction). There are also some examples ofenolate alkylation of cotinine itself (N.-H. Lin et al. (1994) J. Med.Chem., 37, 3542–3553). An interesting preparation of 4′-acetyl-nicotine,as a 1:1 mixture of two epimers, was achieved using a tandem cationicaza-Cope rearrangement-Mannich cyclization reaction starting from aketone (or an aldehyde) and a 2-alkoxy-3-alkenamine (L. E. Overman(1983) J. Am. Chem., Soc., 105, 6622–6629). This reaction can beextended to produce 4′-aldehydo-nicotine, suitable for conjugation.

3-bromo-propylamine hydrobromide (4.2 g) was suspended in 50 mLdichoromethane and triethylamine (about 7 mL) was added until a clearsolution was obtained. The solution was cooled to 0° C. and benzylchloroformate (2.5 mL) was added dropwise. The reaction was allowed toproceed at room temperature for 16 hours under stirring. Theprecipitating salts were filtered off and the clear organic layer waswashed with cold water, cold 1 N HC1 and cold water, dried on sodiumsulfate and evaporated under reduced pressure to a yellow oil (2.93 g ofcrude material).

Cotinine (62 mg) and 3-bromo-N-carbobenzyloxy-propylamine (100 mg) wereseparately co-evaporated with dry toluene. Cotinine was dissolved in 5mL of freshly distilled anhydrous tetrahydrofuran, 60 μL ofN,N,N′,N′-tetramethylenediamine (TMEDA) were added and the solutioncooled to −78° C. by immersion in an ethanol dry ice bath. The cotininesolution was added dropwise to a solution of lithium diisopropylamide(LDA, 200 μL of a 2 M solution in heptane-tetrahydrofuran) intetrahydrofuran, previously cooled to −78° C. The orange mixture isstirred for 15 minutes at −78° C. and then left to warm up in an icebath (2 to 6° C.). The reaction was then cooled again to −78° C. and3-bromo-N-carbobenzyloxy-propylamine dissolve in anhydroustetrahydrofuran added dropwise for 15 minutes. The reaction mixture wasleft to warm-up to −10° C. and then quenched with methanol. The reactionproduct was purified by flash chromatography on a silica gel column.Reduction of the amide of this cotinine derivative was achieved withborane followed by cesium fluoride in hot ethanol. The final amine wasobtained after removal of the carbobenzyloxy group in acidic conditions.

EXAMPLE 3 Synthesis of a Derivitized Nicotine Hapten (Substituted at the5′ Position)

Introduction of a functionalized arm on position 5′ of nicotine can beachieved by reacting appropriately protected alkyl lithium compoundswith cotinine, followed by reduction with sodium cyanoborohydride, inprocedures similar to those described by Shibagaki et al. (1986)Heterocycles, 24, 423–428 and N.-H. Lin et al. (1994) supra.

EXAMPLE 4 Conjugation of a Derivitized Nicotine Hapten to a CarrierProtein

Recombinant exoprotein A (rEPA) is linked to the derivitized nicotinehapten through a succinic acid arm. The 15 lysines of rEPA were readilysuccinylated with succinic anhydride. Then, in a typical conjugationreaction, a 5 to 10 mg/mL solution of the succinylated recombinantexoprotein A (Suc-rEPA) in a 2-(N-morpholino) ethanesulfonic acid (MES)buffer 0.05 M containing 0.15 M NaCl at pH 6.0 was prepared. An equalweight of 3′-aminomethyl-(−)-nicotine (3′AMNic) hapten dissolved in aminimal amount of distilled water was added to the protein solution. ThepH of the hapten solution was adjusted to 6.0 with 0.1 N HCl beforeaddition. Finally, an equal weight of 1-ethyl-3-(3-diethylamino)propylcarbodiimide hydrochloride (EDC) was added to the hapten protein mixtureand the reaction proceeded for 30 min at room temperature whilestirring. The thus obtained nicotine conjugate was purified on aSephadex G-25 column eluted with phosphate buffer saline (PBS) at pH7.4. Conjugate recoveries were in the 80 to 90% range.

EXAMPLE 5 Conjugation of Nicotine-Loaded Matrix

This example describes synthesis of a hapten carrier conjugatecomprising 3′-aminomethyl-(−)-nicotine as a derivitized hapten,recombinant exoprotein A (rEPA) as a carrier protein, adipic aciddihydrazide (ADH) as a linker and poly-L-glutamic acid as a “matrix,” orpolymer support, for the haptens

A poly-L-glutamic acid having an average molecular weight of 39,900 witha polydispersity of 1.15 and a degree of polymerization of 264 was usedin this example. The reacting amounts of hapten and polymer werecalculated so that the target degree of substitution is about 80%. Thatis, when 80% substitution is reached, about 208 hapten units wereconjugated, out of a total 264 repeat units in an average molecule ofthe glutamic acid polymer.

This nicotine-loaded poly-L-glutamic acid has the following formula:

As indicated in the figure, the polyglutamic acid polymer comprisesabout 52 glutamine residues. This number will vary, depending on thebatch and source of the polyamino acid residue chosen for the matrix.Also, the figure indicates that 4 nicotine haptens for each repeatingunit. This number will vary depending on the ratio of reactants usedwhen the matrix and the nicotine hapten are conjugated.

Following conjugation with the derivitized nicotine, the unreactedcarboxylic groups (about 20%) were then derivatized with ADH. When thismatrix was conjugated to a carrier, as described in Example 6, the molarratio of the nicotine-loaded matrix to protein was 1:1. Thus, in thisconjugate, the theoretical nicotine hapten to protein molar ratio wouldbe 200:1, at the completion of the conjugation reaction.

The actual ratio of nicotine substitution on the polyglutamic acid wasestimated using NMR analysis of the product. The intensity of theglutamic acid a-hydrogen peak relative to the four hydrogens of thepyridine ring of the nicotine provide the proportion of nicotineincorporated. The estimated average ratio was 143:1 (nicotine/carrierprotein).

EXAMPLE 6 Preparation of a Nicotine Conjugate Vaccine UsingNicotine-loaded Matrix

A. Loading the Nicotine Hapten on the Matrix

10 mg of poly-L-glutamic acid salt (Sigma, Cat #P4761) were dissolved in2 mL of 0.05 M 2-(N-morpholino)ethansulfonic acid (MES) buffercontaining 0.15 M NaCl at pH 6.0. 10 mg of 3′-aminomethyl-(−)-nicotinewere dissolved in a minimal amount of distilled water and the pH of thesolution was adjusted to pH 6.0 with 0.1N HCl. The nicotine haptensolution was added dropwise to the polypeptide solution while stirringand was subsequently adjust to a pH of 6.0. 20 mg of solid1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) werethen added in three portions to the hapten-polypeptide mixture over aperiod of 20 minutes. The reaction was allowed to proceed for one hourat room temperature. The reaction product (nicotine-substituted matrix)was dialyzed against three changes of water and lyophilized. 12 mg ofnicotine-substituted polyglutamic acid were obtained as a white fluffymaterial.

B. Attachment of the Linker to the Nicotine-Substituted Matrix

10 mg of nicotine-substituted polyglutamic acid were dissolved in 2 mLof MES buffer at pH 6.0. 8 mg of adipic acid dihydrazide (ADH) followedby 10 mg of EDC were added to the solution while stirring. The reactionwas allowed to proceed for 1 hour at room temperature. The obtainedsolution was finally dialyzed against three changes of MES buffer at pH6.0.

C. Conjugation to the Carrier Protein

10 mg of recombinant exoprotein A (rEPA) were dissolved in 2 mL of 0.05M MES buffer at pH 5.6 containing 0.15 NaCl. A volume of ADH-boundnicotine-substituted polyglutamic acid solution estimated to contain 7.5mg of this derivatized material was added to the protein solution. SolidEDC was added to this mixture in three portions over a period of 20minutes while stirring at room temperature. The reaction was allowed toproceed at room temperature overnight. The resulting conjugate wasfinally purified on a Sephadex G-25 column, and eluted with phosphatebuffered saline (PBS) at pH 7.4. This produces a purified preparation ofconjugate, wherein the conjugate contains only the (S)-(−)form ofnicotine.

EXAMPLE 7 Characterization of the Nicotine Carrier Conjugate of Example4

The purified conjugate vaccine of Example 4 was analyzed on a Superose12 size exclusion chromatography column and eluted with PBS at pH 7.4.The hapten to protein molar ratio of 11 to 17 was calculated bydetermining the increase of the UV absorption at 260 nm after theincorporation of nicotine, relative to the absorption at 280 nm. Thisrange was determined by calculating the hapten/carrier protein ratio ofsix separate prepared lots of hapten-carrier conjugate (lot 1: 17.2, lot2: 16.2, lot 3: 13.2; lot 4: 12.0; lot 5: 11.0; lot 6: 17.2). Furtheranalysis to determine this ratio using MALDI-TOF mass spectrometry gaveessentially the same numbers as obtained by UV absorbance difference.The protein concentration of the conjugate vaccine was determined usinga BCA assay. A stability study of the nicotine-carrier conjugate ofexample 4 was carried out. The study used the vaccine vialed in 1 mLglass vials at a concentration of 0.5 mg/mL and the stability of thevialed vaccine was tested at three different temperatures: −70° C., 2 to8° C. and room temperature.

EXAMPLE 8 Stability of the Nicotine Carrier Conjugate of Example 4

The conjugation procedure based on the formation of amide bonds betweenhapten-linker-carrier rather than ester bonds appeared beneficial asobserved in the stability of the conjugate for six months at −70° C., 2to 8° C. and even at room temperature. The stability study consisted ofmonitoring and assaying the conjugate vaccine using the following:

1.) Visual observation to look for any particulates formed (turbidity,precipitation).

2.) Checking for any significant pH change.

3.) Size exclusion chromatography profile in combination with UVabsorption at 260 and 280 nm to determine if the ratio of nicotineincorporation changed.

4.) Reversed phase chromatography to check for any carrier proteindegradation.

5.) SDS PAGE with silver staining looking for any proteolytic cleavageof the conjugated protein.

The conjugation procedure based on the formation of an amide bondbetween the hapten and the linker as well as between the linker and thecarrier appeared beneficial as observed in the stability of theconjugate for six months at −70° C. and 2 to 8° C.

EXAMPLE 9 Evidence of Immunogenicity of the Carrier Conjugate ofExamples 4 and 6

Two nicotine hapten-carrier conjugate vaccines were used to immunizemice, rats, and rabbits.

A. Animal Tests-polyclonal Antibodies

-   -   Animals were immunized using standard protocols. In mice, three        subcutaneous injections of vaccine were administered, two weeks        apart, with test bleeds performed one week following the first        and second injection, and exsanguination occurring one week        following the third injection. Serum samples were evaluated in        an ELISA assay, described in Example 10. The ELISA assay        utilized 3′ AMNic-pGlu bound to microtiter plates.

Rats were immunized intraperitoneally with the vaccines three times.Injections were given two weeks apart with test bleeds performed oneweek following the first and second injection. The rats were thenexsanguinated one week after the third injection. Freund's completeadjuvant was used for the first injection, and incomplete Freund'sadjuvant for the subsequent injections. Serum samples were evaluated inan ELISA assay.

Rabbits were immunized intramuscularly three times, three weeks apartwith 100 μg of vaccine. The initial injection contained Freund'sadjuvant, with subsequent injections containing incomplete Freund'sadjuvant. Rabbits were test bled one week following the second and thirdinjections to ensure adequate titers for production bleeding. Ifadequate titer was acquired as measured by ELISA, rabbits were thenplaced on a weekly production bleed schedule (20 to 40 mL serum perrabbit). Antibody titers were monitored over time and animals wereboosted if necessary to restore antibody levels.

The results of these immunogenicity studies are shown in Tables 1–5.Tables 1 and 2 show the results of an immunogenicity study in mice. InTable 1, the conjugate used was 3′aminomethyl-(−)-nicotine-succinyl-rEPA(Example 4). In Table 2, the conjugate used was3-aminomethyl-(−)-nicotine-polyglutamic acid-ADH-rEPA (Example 6). TheseTables show generation of high titers of antibodies that specificallybind nicotine. Furthermore, these conjugates showed the ability toinduce the booster response.

Tables 3 and 4 show the results of an immunogenicity study in rats. InTable 3, the conjugate used was 3′aminomethyl-(−)-nicotine-succinyl-rEPA(Example 4). In Table 4, the conjugate used was3-aminomethyl-(−)-nicotine-polyglutamic acid-ADH-rEPA (Example 6). TheseTables shown generation of high titers of antibodies that specificallybind nicotine. Furthermore, these conjugates showed the ability toinduce the booster response.

Table 5 shows the results of an immunogenicity study in rabbits. Usingeither 3′aminomethyl-succinyl-rEPA (Example 4) or3-aminomethyl-polyglutamic acid-ADH-rEPA (Example 6), high titers ofantibodies were generated against the two conjugate. Those titersremained elevated for more than 6 months.

TABLE 1 Treatment of mice with 3′AMNic-Suc-rEPA Number of Titer AnimalsDose 1 injection 2 injections 3 injections 10  1 μg 0 36 4,280 10  5 μg1 884 10,727 10 15 μg 3 2,476 14,160 Dose is based on protein assayTiter is the arithmetic mean, one week after corresponding injection.

TABLE 2 Treatment of mice with 3′ AMNic-pGlu-ADH-rEPA Number of TiterAnimals Dose 1 injection 2 injections 3 injections 10  2 μg 2 739 6,58610 10 μg 2 2,490 9,573 10 30 μg 11 2,822 8,195 Dose is based on dryweight of lyophilized conjugate Titer is the arithmetic mean, one weekafter corresponding injection.

TABLE 3 Treatment of rats with 3′ AMNic-Suc-rEPA Number of Titer AnimalsDose 1 injection 2 injections 3 injections 3 15 μg 18 7,942 9,947 3 25μg 4 1,446 5,991 3 50 μg 353 7,211 8,996 Dose is based on protein assayTiter is the arithmetic mean, one week after corresponding injection.

TABLE 4 Treatment of rats with 3′ AMNic-pGlu-ADH-rEPA Number of TiterAnimals Dose 1 injection 2 injections 3 injections 5 100 μg 0 1,0673,752 Dose based on dry weight of lyophilized conjugate Arithmetic mean,one week after corresponding injection

TABLE 5 Treatment of rabbits with 3′AMNic-Suc-rEPA and 3′AMNic-pGlu-ADH-rEPA Immunogen Number of Animals Dose Titer 3′-AMNic-Suc-rEPA 10 100μg 132,000 3′AMNic-pGLu-ADH-rEPA 10 100 μg 147,000 Dose based on proteinassay for 3′AMNic-Suc-rEPA and on dry weight for 3′-AMNic-pGlu-rEPATiter is arithmetic mean, six to seven weeks after third injection

EXAMPLE 10 ELISA Assay and Antibody Specificity

The nicotine molecule itself is not suitable for coating ELISA platesand needs to be linked to a larger molecule having better adhesiveproperties. Poly-L-lysine or poly-L-glutamic acid are commonly used forthis purpose. The derivitized nicotine hapten3′-amimomethyl-(−)-nicotine (3′AMNic) was conjugated to poly-L-glutamicacid and the 3′-aminomethyl-(−)-nicotine-poly-L-glutamic acid conjugate(3′AMNic-pGlu ) obtained was used to coat the ELISA plates.

Antibodies generated against 3′AMNic vaccine were evaluated using a3′AMNic-pGlu ELISA as follows: Dynatech Immulon 4 microtiter plates(Chantilly, Va.) were coated 100 μL/well with 10 ng/mL 3′AMNic-pGlu in0.1 M bicarbonate buffer, pH 9.6 and allowed to incubate overnight (ON)at room temperature (RT). The plates were then aspirated and blockedwith 1% BSA in PBS for one hour at RT. Samples and reference serum werediluted in PBB (1% BSA, 0.3% BRIJ in PBS, pH 7.2) to a dilution whichresults in an approximate optical density (OD) at 450 nm of 2.0. Theplates were washed (9% NaCl, 0.1% BRIJ) five times and diluted samplesand reference serum were loaded. The reference and samples were 2-folddiluted down the plates for a final volume of 100 μL/well and plates areincubated for 1 hour at 37° C. The plates were then washed again andloaded 100 μL/well with peroxidase-conjugated anti-species IgG, Fcspecific (Jackson, West Grove, Pa.) diluted in PBB and incubated at 37°C. for one hour. The plates were washed and incubated 10 minutes at RTwith 100 μl/well 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL,Gaithersburg, Md.) diluted 1:1 with H₂O₂ (supplied with TMB reagentkit). The reaction is stopped with the addition of 100 μL/well 1 Mphosphoric acid and read at 450 nm on an MR4000 microtiter plate reader(Dynatech). Samples are quantified in relation to the reference usingparallel line analysis. The reference is assigned a numerical value(U/mL) that corresponds to the dilution which gives an OD ofapproximately 2.0 at 450 nm.

Antibody specificities were evaluated using an inhibition ELISA assay.Each anti-[3′ AMNic-Suc-rEPA] serum was diluted to a concentration twicethat which would results in an optical density of about 2.0 at 450 nm.Using the 3′ AMNic-pGlu ELISA described above, the diluted antiserum tobe tested was absorbed 1:1 (v/v) with increasing amounts of test antigen(inhibitor) for three hours at 37° C., and that absorbed sample wastested in the ELISA using unabsorbed serum as a reference. Percentabsorption with reference to the unabsorbed sample was determined foreach sample.

The specificity of rat serum containing antibodies raised in response to3′ AMNic-Suc-rEPA, using inhibition ELISA with nicotine tartrate asinhibitor, was calculated. The IC₅₀ for this antibody was 3.5×10⁻⁶ M.The specificity of rabbit serum containing antibodies raised in responseto 3′ AMNic-Suc-rEPA, using inhibition ELISA with nicotine tartrate asinhibitor, was calculated. The IC₅₀ for this antibody was 2.3×10⁻⁵ M.

EXAMPLE 11 Antibody Affinity and Binding Capacity

Antibody binding capacity was measured using equilibrium dialysis for 4hours at 37° C. using 0.7 mL of plasma, Teflon semi-micro cells,Spectrapor 2 membranes with a molecular weight cutoff of 12 to 14 kD andSorenson's buffer (0.13 phosphate, pH 7.4) see Pentel et al., J.Pharmacol. Exp. Ther. 246, 1061–1066 (1987). Plasma pH was measured atthe end of reach equilibrium dialysis run, and samples were used only ifthe final pH is 7.30 to 7.45.

Antibody affinity for nicotine was calculated using a solubleradioimmunoassay, see Mueller, Meth. Enzymol., 92, 589–601 (1983). Themolecular weight of IgG was measured to be 150 kD.

The binding constants and affinities obtained with the radioimmunoassaywere as follows. For anti-[3′ AMNic-Suc-rEPA] rat serum, the IC₅₀(Molar) was 1.36×10⁻⁷. The K_(a)(Molar-1) was 2.57×10⁷. Binding sitesconcentration was 2.61×10⁻⁶ binding sites/L and nicotine-specific IgGconcentration was 0.2 mg/mL.

EXAMPLE 12 Evaluation of Nicotine Distribution in Plasma and Brain ofAnimal Models

The present inventive vaccine has been evaluated in various animalmodels. Rat models were used to determine the effect of active orpassive immunization on nicotine distribution in plasma and brain. Onestudy examined the effects of passive immunization on attenuation of thelocomotor effects of nicotine, which are a central nervous system (CNS)action of nicotine. Another experiment evaluated the effects of passiveimmunization on the effects of nicotine on the cardiovascular system:elevation of the systolic blood pressure.

To evaluate the present immunotherapy, an animal model has beendeveloped to simulate the rapid absorption of nicotine from twocigarettes in humans. This animal model is described in Hieda (1997) J.Pharmacol. Exp. Ther. 283(3):1076–1081. In this model, rats wereadministered 0.03 mg/kg of nicotine by i.v. infusion over 10 sec.,simulating the rapid absorption of nicotine from the lungs in humansmokers. Blood samples were taken at 3 and 10 min after nicotineinjection for measurement of plasma nicotine. When brain nicotineconcentrations were to be determined, animals were sacrificed 3 minafter nicotine injection, and their brains were quickly removed. Thevaccine of example 4 was evaluated in rats to determine its effect onthe distribution of nicotine in plasma and brain.

A. Active Immunization

Rats were immunized with the nicotine vaccine by three i.p. injectionsof 25 μg total per injection of vaccine (3′AMNic-Suc-rEPA) two weeksapart. These animals had increased levels of nicotine in plasma 3 and 10min after an infusion of 0.03 mg/kg of nicotine over 10 seconds,compared with levels in non-immunized controls. See FIG. 1. Thus, activeimmunization was effective in increasing nicotine binding in plasma. Itis known that a modest reduction in the amount of nicotine reaching thebrain can dramatically alter the behavioral effects of nicotine.

B. Passive Immunization

With passive immunization, it was possible to determine the doseresponse effect of immune IgG in increasing plasma nicotine levels andreducing brain nicotine levels. Rats were administered with varyingamounts of anti-(3′AMNic-Suc-rEPA) IgG (12.5 to 50 mg) total perinjection. As shown in FIG. 2, there was a clear dose responseeffect—increasing the dose of IgG increased serum nicotine and decreasedbrain nicotine levels.

FIG. 3 shows that anti-nicotine antibodies (anti-3′AMNic-Suc-rEPA) werepresent and active in the serum of rats, 30 min and 1 day afteradministration of antibodies (50 mg) total per injection. FIG. 3 showsthat following nicotine challenge (0.03 mg/kg infusion over 10 seconds),these antibodies were effective in reducing nicotine concentrations inbrain and increasing nicotine levels in plasma, at 30 minutes and 1 dayafter antibody administration.

Another demonstration of the efficacy of the passive immunization withnicotine vaccine of the invention is its ability to combat consecutiveinfusions of nicotine. In a separate passive immunization experiments inrats, multiple doses of nicotine did not deplete the antibodies presentor significantly reduce their capacity to bind to freshly injectednicotine. In FIG. 4, 50 mg of anti-[3′AMNic-Suc-rEPA] was infused attime zero, 24 hours later, five nicotine injections were made −0.03mg/kg nicotine was infused over 10 seconds, from the right jugular vein,every 20 minutes for 80 minutes. A total of five nicotine injectionswere made. The fifth injection of nicotine was spiked with ³H-nicotine.Total blood and brain were collected 1 minute after the fifth injection.The results are shown below, and are graphically represented in FIGS. 4and 5.

Nicotine Concentrations (mean ± SD) Brain (ng/g) Serum (ng/mL) 5th dose1st dose 5th dose 3H- Nicotine Nicotine 3H-Nicotine Cotinine NicotineNicotine Immune IgG 245 ± 30  343 ± 46  121 ± 17  30 ± 4  244 ± 33  90 ±16 Control IgG 21 ± 3  55 ± 9  41 ± 4  35 ± 5  257 ± 29  126 ± 14  %change +1067 +524 +195 −17 −13 −29

These results show that even after the fifth dose of nicotine, theantibodies are effective in increasing serum nicotine levels, anddecreasing brain nicotine levels. The results with the ³H -nicotinedemonstrate that antibodies are effective against the nicotine injectedat the fifth dose.

EXAMPLE 13 Evaluation of Locomotor Effects of Nicotine

This experiment used was designed to determine whether passiveimmunization could prevent an immediate CNS mediated action of nicotine.The rat model used in this experiment was developed by Dr. David Malinand is described in Malin et al. Nicotine-specific IgG reduceddistribution to brain and attenuates its behavioral and cardiovasculareffects in rats, submitted to the Fifth Annual Meeting of the Societyfor Research on Nicotine and Tobacco, San Diego, Calif., Mar. 5–7, 1999;To establish a baseline, the effect of a subcutaneous injection of 0.8mg/kg of nicotine tartrate on locomotor activity level of rats wasmeasured. The 0.8 mg/kg dose of nicotine tartrate is the highest dosethat could be used without inducing locomotor abnormalities.

There was an increase in activity level after nicotine injection in ratsthat were not pre-treated with anti-[3′AMNic-Suc-rEPA], and in rats thatwere pretreated with 50 mg of normal rabbit serum IgG. See FIG. 6A,right bar and FIG. 6B, left bar. This effect was suppressed bypretreating the animals with 50 mg of anti-[3′AMNic-Suc-rEPA] immune IgG(FIG. 6B, right bar). This shows that the anti-nicotine antiserumeliminated a stimulant effect of nicotine, in vivo.

EXAMPLE 14 Evaluation of Nicotine on Systolic Blood Pressure

In this experiment, another indicator of the behavioral effect ofnicotine was measured: the change in systolic blood pressure. Rats werepretreated with anti-[3′AMNic-Suc-rEPA] IgG, or control IgG. Rats weretreated with a subcutaneous injection of 0.1 mg/kg nicotine tartrate.Control rats showed an increase in systolic blood pressure of 42.6±3.2mm Hg, when treated with nicotine. When rats were pretreated withanti-nicotine antiserum IgG, the nicotine challenge was less effective.When increasing amounts of anti-nicotine serum were administered, thisdiminished the ability of nicotine to raise blood pressure. As shown inFIG. 7, there was a negative linear trend of blood pressure as afunction of IgG dose.

1. A nicotine hapten of formula II:

wherein n is 1 to 12 and Z is NH₂, COOH, CHO or SH and —(CH₂)_(n)—Z canbe bonded to the 3′, 4′ or 5′ position.
 2. A vaccine compositioncomprising at least one nicotine hapten of claim 1.